Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Linker histone H1 is essential for Drosophila development, the establishment of pericentric heterochromatin, and a normal polytene chromosome structure Xingwu Lu, Sandeep N. Wontakal, Alexander V. Emelyanov, Patrick Morcillo,1 Alexander Y. Konev,2 Dmitry V. Fyodorov,3 and Arthur I. Skoultchi4 Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA We generated mutant alleles of Drosophila melanogaster in which expression of the linker histone H1 can be down-regulated over a wide range by RNAi. When the H1 protein level is reduced to ;20% of the level in wild- type larvae, lethality occurs in the late larval – pupal stages of development. Here we show that H1 has an important function in gene regulation within or near heterochromatin. It is a strong dominant suppressor of position effect variegation (PEV). Similar to other suppressors of PEV, H1 is simultaneously involved in both the repression of euchromatic genes brought to the vicinity of pericentric heterochromatin and the activation of heterochromatic genes that depend on their pericentric localization for maximal transcriptional activity. Studies of H1-depleted salivary gland polytene chromosomes show that H1 participates in several fundamental aspects of chromosome structure and function. First, H1 is required for heterochromatin structural integrity and the deposition or maintenance of major pericentric heterochromatin-associated histone marks, including H3K9Me2 and H4K20Me2. Second, H1 also plays an unexpected role in the alignment of endoreplicated sister chromatids. Finally, H1 is essential for organization of pericentric regions of all polytene chromosomes into a single chromocenter. Thus, linker histone H1 is essential in Drosophila and plays a fundamental role in the architecture and activity of chromosomes in vivo. [Keywords: Linker histone H1; heterochromatin; histone methylation; polytene chromosomes; chromocenter; position effect variegation] Supplemental material is available at http://www.genesdev.org. Received October 6, 2008; revised version accepted January 5, 2009. The genomes of eukaryotes are packaged into a highly around which ;145 base pairs (bp) of DNA are wrapped compact nucleoprotein complex called chromatin. The and protected from nuclease digestion (Van Holde 1988; histones constitute a family of proteins that are inti- Wolffe 1998). The linker histone H1 binds to core mately involved in organizing chromatin structure. particles and protects an additional ;20 bp of DNA There are five major classes of histones: the core histones (linker DNA). In metazoans, the abundance of linker H2A, H2B, H3, and H4, and the linker histones usually histones, although variable during development, ap- referred to as H1. The nucleosome core particle is the proaches that of core histones (Woodcock et al. 2006), highly conserved repetitive unit of chromatin organiza- suggesting that they play an important role in establish- tion. It consists of an octamer of the four core histones ing and maintaining the structure of the chromatin fiber. Much of our knowledge about the roles of linker histones Present addresses: 1Departamento de Gene´tica, Universidad de Valencia, comes from in vitro studies. These studies indicate that Doctor Moliner 50, E46100 Burjassot, Valencia, Spain; 2Molecular and two principal functions of linker histones are to stabilize Radiation Biophysics Department, St. Petersburgh Nuclear Physics In- the DNA entering and exiting the core particle and to stitute, Gatchina 188300, Russia. Corresponding authors. facilitate the folding of nucleosome arrays into more 3E-MAIL [email protected]; FAX (718) 430-8574. compact structures (Ramakrishnan 1997; Wolffe 1997). 4E-MAIL [email protected]; FAX (718) 430-8574. Article published online ahead of print. Article and publication date are H1 also affects nucleosome core particle spacing and online at http://www.genesdev.org/cgi/doi/10.1101/gad.1749309. mobility. In vitro studies also suggest that H1 acts primarily 452 GENES & DEVELOPMENT 23:452–465 Ó 2009 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/09; www.genesdev.org Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Drosophila histone H1 function in vivo as a transcriptional repressor (Laybourn and Kadonaga Results 1991; Brown et al. 1996). Recent attempts to study the Linker histone H1 is essential for Drosophila functions of linker histones in vivo have used gene development inactivation approaches. Elimination of the linker his- tone-like protein Hho1p in Saccharomyces cerevisiae did To deplete H1 histone in vivo in Drosophila, we used not cause any major phenotypic effects, nor were any RNAi. The H1 RNAi expression vector is depicted in perturbations in chromosome structure apparent (Ushinsky Figure 1A. It was constructed by inserting PCR fragments et al. 1997; Patterton et al. 1998; Hellauer et al. 2001; encompassing the first 600 bp of the Drosophila mela- Downs et al. 2003). This finding may be explained by the nogaster H1 coding sequence (encoding the first 200 much lower abundance of Hho1p in yeast (1:37 core amino acids of the 256-amino-acid full-length H1 protein) particles). The absence of any phenotypic effects was also in opposite orientations on both sides of the first intron of reported upon elimination of H1 from Aspergillus nidu- the actin 5C gene in pINT-1 (Wei et al. 2007). pINT-1 is lans, which has a more typical linker histone (Ramon a modified pUAST vector in which transcription is driven et al. 2000). H1 also was found to be dispensable for by the GAL4-responsive UAS promoter. We prepared six growth and viability of Tetrahymena (Shen et al. 1995). individual pINT-1-H1 transgenic lines, and homozygous However, its elimination by gene inactivation led to transgenic flies were mated at various temperatures to partially decondensed chromatin, supporting an in vivo counterparts of the opposite sex that carry the Tubulin- role for H1 in chromatin folding. GAL4 driver (TubTGAL4/TM3, Sb) (see the Materials and Mammals express at least eight nonallelic H1 subtypes Methods). Another pINT-1 transgenic line (pINT-1-Nau) that differ in their expression during development. Al- (Wei et al. 2007) encoding a dsRNA for D. melanogaster though none of the eight individual subtypes appear to Nautilus protein was used as a control. In the adult be essential, mouse embryos in which the stoichiometry offspring of the experimental cross at 29°C, we did not of H1 to the core particles has been reduced ;50% by observe flies that carry both the pINT-1-H1 and Tubulin- inactivation of three of the five somatic linker histone GAL4 transgenes, and we observed few offspring at 26°C. subtype genes die at midgestation. This observation At the same time, expected numbers of flies with pINT-1- indicates that the total amount of linker histones is Nau and Tubulin-GAL4 transgenes were obtained at both important for normal mammalian development (Fan temperatures (Table 1). Thus, the expression of H1- et al. 2003). In the knockout embryos and embryonic specific dsRNA under control of the ubiquitous tubulin stem (ES) cells derived from them, H1 is an important promoter causes lethality before eclosion. determinant of nucleosome spacing and of local chroma- To confirm that the combination of the pINT-1-H1 and tin folding in vivo (Fan et al. 2005). However, despite Tubulin-GAL4 transgenes causes depletion of H1 histone these advances, the role of H1 in higher-order chromatin protein, larvae of this genotype were collected at 29°C, folding and long-range chromosome structure remains salivary glands were dissected, and tissue lysates were enigmatic. examined by SDS-PAGE and immunoblotting with his- Further advances in our understanding of linker his- tone H1 antiserum. We observed that lysates from pINT- tone functions would be greatly facilitated by studies in 1-H15M + Tubulin-GAL4 salivary glands contained <5% a genetically tractable organism where H1 may prove to of the H1 protein present in control lysates (Fig. 1B). We play an essential role. As mentioned, deletion of the yeast also determined the extent of H1 depletion in animals HHO1 gene does not lead to obvious phenotypic effects. with other H1 RNAi transgene insertions at various Although linker histones are essential for embryonic temperatures (Supplemental Fig. 1; Supplemental Table development in mice, the existence of multiple, non- 1). Depending on the temperature and the insertion allele, allelic mouse H1 variant genes, as well as apparent the expression of H1 protein in salivary glands of trans- compensatory gene expression mechanisms within this genic larvae was decreased by 14%–95% in salivary gene family, has hindered attempts to study the effects of glands and by 13%–99% in whole larvae. GAL4 is known decreasing H1 expression in this species (Fan et al. 2005). to exert higher transcriptional activity at elevated tem- On the other hand, Drosophila offers an attractive alter- peratures (Brand et al. 1994). Consistent with this obser- native for such studies because it contains a single linker vation, the lethal effect of combining the pINT-1-H1 histone protein. However, it is encoded by ;100 copies of and Tubulin-GAL4 transgenes was alleviated at lower the H1 gene positioned within a tandemly repeated array temperatures (18°C and 22°C), due to reduced dosage of five sequentially arranged histone genes (Lifton et al. of the H1-specific siRNA (Table 1; Supplemental Fig. 1978). Thus, specific elimination of Drosophila H1 his- 1; Supplemental Table 1). It appears from our analyses tone by classical genetic approaches is not feasible. Here that lethality is caused by the knockdown when H1 we describe the use of specific RNAi to nearly completely protein levels are decreased below a certain threshold, deplete H1 in Drosophila in vivo. This approach has between ;20% and 60% of the wild-type level (Supple- allowed us to demonstrate that the H1 linker histone is mental Table 1).